Methods for the detection of biologically relevant molecules and their interaction characteristics

ABSTRACT

Methods for the detection of biologically relevant molecules that comprise concentrating such molecules into microscopic holes in a sheet of chemically inert material, restricting the openings, and measuring the electric current through the holes or the fluorescence near the hole openings. The electric current or fluorescence will change as the molecules diffuse out of the holes, providing a measure of the diffusion rate and thereby detecting the presence and characteristics of the molecules. For molecules that interact, the diffusion rate will be slower than for molecules that do not interact, yielding a determination of the molecular interaction. Capping the population of holes and inserting into a mass spectrometer allows identification of the molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/498,141, having an international filing date of 15 Feb. 2011, nowallowed, which is the National Stage Application of PCT/US2011/024882,having an international filing date of 15 Feb. 2011, which claimsbenefit under 35 U.S.C. Section 119(e) of U.S. Provisional PatentApplication No. 61/338,676, filed 23 Feb. 2010. The contents of theabove patent applications are incorporated by reference herein in theirentirety.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to methods for the detection of analyteparticles, and determining their interaction characteristics withincomplex media. More particularly, this invention relates to diffusionimmunoassays and their functional equivalents.

2. Description of the Related Art

Biologically relevant molecules, such as proteins and nucleic acids, arecommonly associated with biological systems, where they form a complexnetwork of interactions for the performance of tasks such as cellreplication, metabolism, self-regulation, intercellular signaling, andimmune response. Diseases distort this network, and understanding thisdistortion is fundamental to early detection of disease and chemicalrepair of the distortion through drug therapy. There are a number ofexisting techniques for identifying and characterizing these largemolecules to gain an understanding of the interaction network, but eachsuffers from particular limitations. Techniques used for nucleic acids,such as DNA, have been largely successful due to their analyticallyfavorable properties, but proteins are chemically and physicallydiverse. This diversity results in analytical techniques that are bynecessity narrowly focused, when a broad technique would be much morehelpful in characterizing a complex protein network. Furthermore,proteins may be functionally significant at even undetectableconcentrations, yet cannot be amplified with the ease of nucleic acids,necessitating techniques that have a high intrinsic sensitivity.

Genetic Methods

Protein interactions can be investigated by using classical genetics.Different mutations are combined into in the same cell or organism, andthen the resulting phenotype is observed. This ensures that proteininteractions occur in their near perfectly native environments,Unfortunately, these methods are applicable only to a small group ofproteins, and can not be used for exploring the whole proteome.Furthermore, phenotypic changes can be caused for a multitude of reasonsrelated to the gene mutations, and thus protein interactions suggestedby experimental results would require confirmation at the biochemicallevel.

Bioinformatic Methods

Protein interactions can be investigated by using comparative genomicsfor the functional annotation of proteins. Currently, there are threemajor techniques. The first technique is called Domain Fusion (orRosetta Stone), which assumes that protein domains are structurally andfunctionally independent units that can operate as discretepolypeptides. The second technique is based on the operon organizationof bacterial genes, where such genes are often functionally related evenif their actual sequences are disparate. The third technique usesphylogenic profiling, exploiting the evolutionary conservation of genesinvolved together in a particular function. Unfortunately, thesebioinformatic methods require a complete genome sequence, and aregenerally limited to bacteria or other organisms with well-definedoperons. Furthermore, the results are not conclusive evidence ofspecific protein interactions, and require confirmation at thebiochemical level.

Affinity-Based Methods

Protein interactions can be investigated at the biochemical level bydirectly determining affinity between a protein and candidateinteraction partners, such as in immunoassays. Proteins are immobilizedonto a stationary phase or flat glass surface, and a mixture ofpotential complementary ligands is flooded over the immobilized protein.Binding is indicated by fluorescent or radioactive probes chemicallyattached to the ligands, which are then imaged. Unfortunately, proteinfunctionality can be severely restricted by the immobilization process.A related technique chemically labels the proteins themselves and thenfloods them over a surface coated with immobilized ligands. However,this process suffers from the fact that proteins do not label uniformlywith the same efficiency, and the chemical attachment of the labels caninterfere with the range of the protein's interactions. Furthermore,attachment of labels can adversely affect protein solubility, andfluorescent probes may be quenched by the attachment. Detection may alsobe performed by electrochemical amperometry (e.g. U.S. Pat. No.7,297,312), but the drawbacks remain.

Diffusion Immunoassays may use a pair of adjacent fluid flows in amicrocapillary channel (e.g. U.S. Pat. No. 6,541,213, U.S. Pat. No.7,271,007, U.S. Pat. No. 7,306,672, U.S. Pat. No. 7,060,446, U.S. Pat.No. 7,704,322, and U.S. patent applications US 2010/0263732, US2008/0182273, US 2003/0096310, US 2009/0053732, and US 2003/0234356), orfunctional equivalents, where interactions between components in the twofluids at the flow interface causes a change in diffusioncharacteristics that affects the concentration profile near fluidinterface. Detection of the concentration profile provides informationon the interactions. This avoids complications associated with astationary phase, but still prefers the use of labeling, and only onemeasurement per sample is practical it is non-cyclable). The use ofmultivalent reactants (e.g. U.S. Pat. No. 7,550,267) allows the use ofcomponents with a greater disparity of diffusion coefficients, but themeasurement drawbacks of labeling and non-cyclability remain. Relateddevices using porous membranes (e.g. U.S. Pat. No. 5,212,065) sufferfrom the same disadvantages. The use of a thin polymer layer over anarray of electrochemical sensors (e.g. U.S. Pat. No. 7,144,553) iscapable of determining diffusion characteristics via time delaysinvolved in permeating the polymer, but does not concentrate the analytein a narrow hole (thereby enhancing sensitivity), and is not amenable tocycling the analyte towards and away from the sensor via hydrodynamicflow. Diffusion may be measured by optically tracking an analyte system(e.g. US 2008/0145856), but this has the drawback of preferring the useof labeling technology. Diffusion may be measured by detection ofpenetration depth into a hydrogel (e.g. US 2006/0115905), but this hasthe drawback of preferring the use of labeling technology.

Microchannel Conductometry measures changes to transverse conductance asprotein molecules pass through a microchannel, and this has beendescribed as possibly useful for label-free protein interactiondetection (e.g. US 2005/0109621). However, that method only indirectlydetermines diffusion properties, and is not amenable cycling themeasurements. Conductometry has also been used for label-free cellculture monitoring (e.g., U.S. Pat. No. 7,732,127 and U.S. Pat. No.7,192,752), but these are not direct measurements of proteins and theirinteractions. The use of nanogaps US 2005/0136419) avoids certaindouble-layer complications of electrochemical measurements, but has thedrawback of preferring the use of tethering technology.

Physical Methods

Protein interactions can be investigated at the physical level. Thetechniques of X-ray crystallography and nuclear magnetic resonance (NMR)determine the locations of protein atoms within the molecule, and theresulting 3-dimensional map can be used to suggest which other moleculesare likely to fit into its topology and charge distribution.Unfortunately, X-ray crystallography requires the growth of proteincrystals for each protein to be investigated, which is a difficult andtime consuming process, and the crystal environment is drasticallydifferent than the aqueous environment in which the protein functions.NMR requires a large quantity of purified protein, and analysis of theresulting complex data can be inconclusive.

The technique of surface plasmon resonance uses protein adherence tometal films, but this can adversely affect protein functionality.

The technique of Fluorescence Resonance Energy Transfer (FRET) takesadvantage of energy transfer that can occur between nearby fluorophoreswhen the emission spectrum of one fluorophore overlaps the excitation ofthe other fluorophore. By labeling one candidate interaction partnerwith one fluorophore, and the other candidate interaction partner withanother fluorophore, then interactions will be indicated by an increasein the fluorescence of one fluorophore at the expense of the other. Thisworks well with even transient interactions. Unfortunately, thisrequires chemical attachment of a fluorophore to every protein, whichmay adversely affect protein functionality.

The technique of atomic force microscopy of dendron-isolated analytes(e.g. U.S. Pat. No. 6,645,558, US 2008/0113353, US 2009/0048120 and US2010/0261615) can detect individual analyte molecules, but requirestethering bonds and extensive sample preparation.

Standard Expression Libraries

Protein interactions can be investigated through the use of libraries ofcDNA that produce bait proteins that can be labeled and used as a probe.Typically, the bait proteins are produced through the use of phageparticles. The technique allows for the association of as bait proteinwith its corresponding cDNA, but suffers from the major drawback of aslow throughput; screenings for each bait protein are required.Furthermore, the production of the bait proteins is not under nativeconditions, leading to possibly erroneous folding and false negatives.

Phage Interaction Display

Protein interactions can be investigated through the use of anexpression cloning strategy. A cDNA sequence is inserted into a phageprotein coat gene, and cultured in bacterial cells. The phage thanexpresses as new protein on its coat, which then can be used for proteininteraction analyses. If a mixture of such phages interacts with animmobilized labeled protein in a well, the well can be rinsed to leavebehind only the interacting phages, along with the cDNA sequences thatformed them. The cDNA sequences in turn can then be massively amplifiedby bacterial infection. This technique is highly amenable to automatedparallel screenings. Unfortunately, as with standard expressionlibraries, the proteins are not formed under native conditions. Also,the technique is limited to short peptides that can be formed on thephage surface.

Yeast Two-Hybrid System

Protein interactions can be investigated through the use oftranscription factors within yeast cells, which is a more nativeenvironment for protein expression than in vitro. A protein underinvestigation is expressed in a haploid yeast cell as a fusion with theDNA-binding domain from a transcription factor. Another protein isexpressed in another haploid yeast cell as a fusion with thetransactivation domain of the same transcription factor. Mating the twoyeast strains into a diploid strain allows the two proteins to interact.If they do interact, the transcription factor will be assembled, causinga test gene to be activated. The technique is amenable to large-scalescreenings, but there are several drawbacks. Experimental repeatabilityis quite low, suggesting inordinate sensitivity to environmentalconditions, or that the screens were not comprehensive. There are asignificant number of failures to detect interactions well-establishedfrom other more specific techniques, indicating high level of falsenegatives. Lastly, a significant number of detected interactions aredetermined to not be valid by further analysis, indicating a high levelof false positives.

All publications referred to herein are hereby incorporated by referencein their entirety to the extent not inconsistent herewith.

SUMMARY OF THE INVENTION

The methods described herein use a combination of existing technologiescomprising spheroids, magnetic fields, fluorescence, optics, filtertechnology, gel technology, chemistry, electrochemistry, chromatography,and Matrix-Assisted Laser Desorption Ionization (MALDI), to detect andcharacterize analyte particles (e.g. biologically relevant molecules),to characterize any structural changes to analyte particles, and toidentify their interactions.

In one method in accordance with the invention, analyte particles aretrapped within microscopic reservoirs in an ionic environment (such asaqueous saline), by means such as solvent flow or an electric field.Once trapped, the means of trapping is terminated, and the electricalconductivity of the reservoir is measured as the analyte particlesdiffuse out of the reservoirs. The electrical conductivity is a measureof the ease of ion flow. Since the ion flow is restricted by the analyteparticles, the conductivity will drop as the analyte particles diffuseout of the reservoirs. The time frame over which the Conductivity dropsprovides a measure of the analyte particle diffusion coefficient. Thediffusion coefficient in turn provides a measure of the analyte particlecharacteristics, such as its size and shape. The use of an electricfield to supplement or suppress the diffusion can also be used to studythe charge characteristics of the analyte particle. Furthermore, twoanalyte particles that exhibit a binding interaction will display areduction in the diffusion coefficient relative to the individualanalyte particles; the reduction provides a measure of the bindinginteraction. After the analyte particles have diffused out of thereservoirs, they may be trapped again within the reservoirs, so that themeasurement can be repeated in a continual cycle.

In another method that is similar to the first method, fluorescence isused instead of electrical conductivity to provide a measure of theanalyte particle diffusion coefficient. The fluorescence can be usedeither by causing the restriction of ion flow in an electric field todelay the onset of fluorescence, or by causing restriction of the flowof fluorescent particles themselves.

Further methods can involve physically trapping the analyte particleswithin the reservoirs, so that the reservoirs can be moved en masse intoa vacuum chamber of a mass spectrometer while remaining in an aqueousenvironment. Laser ablation can then be used to vaporize the aqueousmatrix of the analyte particles to provide a mass spectrometry samplewith improved control over the molecular fragmentation.

The methods of the invention can be used for finding binary, ternary, orgreater interactions, for analyte particles having a large sizedifference, and for situations where one or more of the participatinganalyte particles are in a lipid environment. Such methods may findwidespread applicability for biomarker discovery, drug discovery, anddrug evaluation. A strong advantage of these methods over existingmethods is that it is label-free and tether-free, ensuring that analyteparticles interact in their native state without chemically attachedlabels or tethers; labels and tethers may still be used, but they arenot required. The methods are also largely independent of pH or othersolution characteristics, can be used with opaque complex aqueousmixtures, can use extremely small sample volumes, and allow themeasurements to be cycled (i.e. repeated) for enhanced sensitivity.

In accordance with one aspect of the invention a method for thedetection of analyte particle presence, characteristics, andinteractions, comprises: providing a sheet of material having aplurality of through holes that are of substantially similar diameter;restricting the hole openings of at least one face of the material;inserting analyte particles into a sub-population of said holes;applying an electric field through said through holes containing theanalyte particles; measuring a change in electric current flow with timeindicative of the diffusion rates of said analyte particles; and whereinthe diffusion rates of said analyte particles provide a measure ofanalyte particle presence, characteristics, and interaction.

The sheet of material can comprise a polycarbonate; a track-etchedpolycarbonate; a polymer drilled with a plurality of holes; a polymerchemically etched with a plurality of holes; a glass drilled with aplurality of holes; a glass chemically etched with a plurality of holes;a perforated polymer film; a perforated monolayer film or a perforatedmultilayer film. Typically the material is electrically insulating andis substantially chemically inert. The sheet of material can be of athickness in as range 500 nm to 1000000 nm. Alternatively it is of athickness in a range 1 nm to 10 cm. The through holes can be of diameter10 nm to 5000 nm. Alternatively the through holes can be of diameter 1nm to 1 cm. In one arrangement an inner surface of the through holes ischemically derivatized. Alternatively an outer surface of the throughholes can be chemically derivatized. The through holes can be filledwith a gel.

In one embodiment the hole openings are restricted by applying a layerof gel in contact with a surface of said sheet of material. The gel cancomprise a gelatin; an agarose; a polyacrylamide; a polyacylate; apermeable polymer; a permeable copolymer; a starch; an aerogel; acollodion; a dialysis membrane; a fluid immiscible with the analyteparticle matrix; any of the above-listed materials in a chemicallymodified form; any of the above-listed materials embedded with particlesand combinations thereof.

In another embodiment the hole openings are restricted using spheroidshaving a diameter configured to cause substantial restriction to fluidflow through the holes. The spheroids can be held in position bygravity; centripetal force; centrifugal force; hydrodynamic pressure;hydrostatic pressure; chemical bonds or using a gel matrix. Depending onthe composition of the spheroids the spheroids can be held in positionby applying an electric field or by applying a magnetic field gradient.

Where the spheroids are held in position using a gel matrix the methodcan further comprise removing the spheroids from the gel matrix.

Advantageously the electric current flow is measured using anamperometer that is configured to measure the electric current through aselected area of the sheet, at a rate sufficient for the diffusion ratesbeing measured.

The said selected area can be selected using an insulating tube with oneend in physical contact with the sheet of material; an insulating sheetwith a hole that is applied to the surface of said sheet of material oran insulating water-immiscible fluid that is applied to the surface ofthe sheet of material.

According to another aspect of the invention a method for the detectionof analyte particle presence, characteristics, and interactions,comprises: providing a sheet of material having a plurality of throughholes that are of substantially similar diameter; restricting the holeopenings of at least one face of the material; inserting analyteparticles into a sub-population of said holes; passing a migration forceaxially through said through holes containing the analyte particles:measuring a change in fluorescence with time indicative of the diffusionrates of said analyte particles; and wherein the diffusion lilacs ofsaid analyte particles provide a measure of analyte particle presence,characteristics, and interaction.

Preferably fluorescence is measured by a photometric system capable ofmeasuring the fluorescence of a selected area of the sheet, at a ratesufficient for the diffusion rates being measured.

According to a further aspect of the invention a method foridentification of analyte particles, comprises: providing a sheet ofmaterial having a plurality of through holes that are of substantiallysimilar diameter; restricting the hole openings of at least one face ofthe material; inserting analyte particles into a sub-population of saidholes; closing substantially said hole openings; inserting said sheet ofmaterial into a mass spectrometer; ablating a selected area of saidsheet of material; ionizing the resulting products of measuring themass/charge ratios of the resulting ions; and wherein the mass/chargeratios provide a means for identification of the analyte particles.

The method can further comprise restricting the hole openings bycompressing spheroids into the entrances of said hole openings such thatsaid openings are substantially closed.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the present invention is better understood, methods inaccordance with the invention will now be described, by way of exampleonly, with reference to the accompanying drawings in which:

FIG. 1A is schematic of a perforated material used in the method of theinvention;

FIG. 1B is a sectional side view of the perforated material of FIG. 1Athrough a line x-x;

FIG. 2 is a magnified view of a single hole in the perforated materialof FIG. 1;

FIG. 3 is an illustration of the manufacture of a bilayer material ofcontrolled thickness;

FIG. 4 is an illustration of the known chemistry for formingpolyacrylamide gel;

FIG. 5 is a magnified view of the bilayer material from FIG. 3;

FIG. 6 is an illustration of the known chemistry of peptide bondsynthesis;

FIG. 7 is a schematic of both the perforated material of FIG. 1 and thebilayer material of FIG. 3, in close proximity;

FIG. 8 is a schematic of a structure resulting from the perforatedmaterial of FIG. 1 and the bilayer material of FIG. 3 being boundtogether by peptide bonds;

FIG. 9 is a schematic of the structure of FIG. 8, with the outer layerof the bilayer removed, leaving a layer of gel adhered to the perforatedmaterial;

FIG. 10 is a schematic of a variation in FIG. 9, having fluorophores orsurfactants chemically bound to the outer surface of the gel;

FIG. 11 is a schematic of a variation of FIG. 9, having a spheroidclogging one end of a hole in the perforated material before assembly ofthe structure, with a small gap;

FIG. 12 is a schematic of the unclogging effect of a force, such as froma magnetic field gradient, by moving the position of the spheroid ofFIG. 11;

FIG. 13 is a schematic of a variation of FIG. 9, having a spheroidclogging one end of a hole in the perforated material before assembly ofthe structure, with no gap;

FIG. 14 is a schematic of a variation of FIG. 9, having spheroidsclogging both ends of a hole in the perforated material;

FIG. 15 is a schematic of as tear in the gel resulting from removal ofone of the spheroids of FIG. 14;

FIG. 16 is a schematic of a variation of FIG. 9, having a spheroidclogging one end of a hole in the perforated material, with no gel, heldin place by a force, such as from a magnetic field gradient;

FIG. 17 is a schematic of fluid flow through the hole of the structureof FIG. 9;

FIG. 18 is a schematic of the accumulation of analyte particles in thehole of the structure of FIG. 9;

FIG. 19 is a schematic of fluorophore migration induced by an electricfield E;

FIG. 20 is a schematic of fluorophore migration induced by as magneticfield gradient nabla B;

FIG. 21 is a schematic of fluorophore migration induced by agravitational field G;

FIG. 22 is a schematic of electrolyte migration induced by an electricfield E;

FIG. 23 is a schematic of capacitor formation resulting from electrolytemigration induced by an electric field E;

FIG. 24 is an illustration of the electric field associated with thecapacitor of FIG. 23;

FIG. 25 is an illustration of the transfer of electric charge to thefluorophore of FIG. 23;

FIG. 26 is a schematic of fluorophore migration around molecularobstacles for a) no obstacles b) small obstacles and c) substantialobstacles;

FIG. 27 is a schematic of electrolyte migration around molecularobstacles for a) no obstacles b) small obstacles and c) substantialobstacles;

FIG. 28 is a schematic of electrolyte migration around charged molecularobstacles for a) no obstacles b) small obstacles and c) substantialobstacles;

FIG. 29 is a schematic of excitation of migrated fluorophores withultraviolet light, and their emission of visible light;

FIG. 30 is a schematic of excitation of charged fluorophores withultraviolet light, and their emission of visible light;

FIG. 31 is a schematic of electric current flow to in electrode;

FIG. 32 is a schematic of how analyte particles are loaded into theholes of the structure of FIG. 9;

FIG. 33A is a schematic of how electric current is detected in ananalytically useful way;

FIG. 33B is an expanded view of the tip of the tube that deliversanalyte particles in FIG. 33A;

FIG. 33C is a view of the conductance map resulting from FIG. 33A;

FIG. 34A is a schematic of how fluorescence is detected in ananalytically useful way;

FIG. 34B is a view of the photographic image resulting from FIG. 34A;

FIG. 35 is an illustration of the conductance maps that evolve over timewhen the electric field is applied;

FIG. 36 is an illustration of the photographic images that evolve overtime when the migration force is applied;

FIG. 37 is an illustration of a vertical (increasing) shift in theelectric current for a small grouping of holes, protein molecules absentversus protein molecules present;

FIG. 38 is an illustration of a horizontal (temporal) shift in thefluorescence inflection point for a small grouping of holes, proteinmolecules absent versus protein molecules present;

FIG. 39 is an illustration of repeated delta current measurement of thepresence of protein molecules in a small grouping of holes, and theabsence of protein molecules in another small grouping of holes;

FIG. 40 is an illustration of repeated delta time measurement of thepresence of protein molecules in a small grouping of holes, and theabsence of protein molecules in another small grouping of holes;

FIG. 41 is an illustration of repeated delta current measurement ofprotein binary interaction in a small grouping of holes, and of proteinbinary non-interaction in another small grouping of holes;

FIG. 42 is an illustration of repeated delta time measurement of proteinbinary interaction in a small grouping of holes, and of protein binarynon-interaction in another small grouping of holes;

FIG. 43 is a schematic of a large population of holes of the perforatedmaterial of FIG. 9;

FIG. 44 is a schematic of a first stage of permanent entrapment ofmolecular species in the perforated material;

FIG. 45 is a schematic of a second stage of permanent entrapment ofmolecular species in the perforated material;

FIG. 46 is a schematic of permanently trapped molecular species in theperforated material;

FIG. 47 is a schematic of illumination by a laser of trapped molecularspecies in a hole of the perforated material; and

FIG. 48 is a schematic of shows vaporization of the trapped molecularspecies of FIG. 43.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention use a combination of existing technologiesin a unique manner. These existing technologies comprise spheroids,magnetic fields, fluorescence, optics, filter technology, geltechnology, chemistry, electrochemistry, chromatography, andMatrix-Assisted Laser Desorption Ionization (MALDI). These will beoverviewed individually, and then methods in accordance with variousembodiments of the invention will be described. Throughout this patentspecification like reference numerals are used to denote like parts.

Spheroids

Spheroids are commercially available from a wide array of manufacturers,such as from: http://www.microspheres-nanospheres.com/

They have a high-precision diameters ranging from 50 nm up to 5000 nm,and can be made out various materials such as polystyrene orpolystyrene/melamine copolymer. Additionally, they can be impregnatedwith magnetically active materials, and impregnated with fluorescentmaterials. Furthermore, these particles can be manufactured with asderivatized surface, such as amine (NH2) or carboxyl (COOH), to whichmany ligands can be attached with well-established chemistry. They arecommonly used to protein purification procedures, where a particularprotein binds to the surface ligands, and then can be magneticallyextracted from bulk solution.

Magnetic Fields

In a magnetic field gradient, a particle having a magnetic moment, suchas a particle of ferrous oxide, will experience a force directed towardsthe convergence of the field, with a magnitude that is simply theproduct of the magnetic moment M and the intensity of the gradient nablaB.

A magnetic field gradient can be generated by a simple solenoid carryingan electric current. Typical solenoids have the point of maximummagnetic field gradient just outside the ends of the solenoid, alwaysdirected towards the center of the solenoid. A pair of opposingsolenoids, each carrying an offset sine wave of electric current, can beused to generate a magnetic field gradient of arbitrary strength andpolarity.

Fluorescence

Fluorescent materials absorb light of a short wavelength, and thenrelease the energy as light of a longer wavelength. There is a verylarge array of commercially available fluorescent dyes that findwidespread use in the biotechnology field. One example is sodiumfluoresceinate, which absorbs ultraviolet light and emits a green-yellowlight. The neutral form, fluorescein, is non-fluorescent. In addition tofluorescent molecules, a newer type of material called Quantum Dots arealso fluorescent. They are composed of particles of a semiconductor,such as cadmium sulphide (CdS) or cadmium telluride (CdTe). They arevery small, of the order of a few nanometers in diameter, and areextremely fluorescent. The surface of the particles can be derivatizedwith various materials, such as amine (NH2) or carboxyl (COOH), to whichmany ligands can be attached with well-established chemistry. They arecommonly used for microscopy stains.

Optics

Fluorescence can be detected by exciting the fluorescence with shortwavelength light (such as ultraviolet light), and collecting the emittedlonger wavelength light (such as visible light) with a photosensor.Examples of ultraviolet light sources are mercury-vapor bulbs and LEDlasers. For near-ultraviolet, the 405 nm LED lasers commonly used forBlu-Ray disks are particularly convenient. The emitted fluorescence maybe collected with optical lenses or light pipes and directed into aphotosensor. Examples of photosensors are photomultiplier tubes anddigital cameras. Photomultiplier tubes have high sensitivity and speed,and digital cameras can measure broad areas.

Filter Technology

A unique type of perforated material 10 comprising track-etchedpolycarbonate (TEPC) is commercially available for use in filtering,such as from Millipore Corp. or Whatman Corp.

TEPC comprises a sheet of polycarbonate material 12 having a smooth,glass-like surface, randomly punctured by very uniformly sized throughholes 14. FIGS. 1A and 1B are schematic representations of such aperforated material 10. These holes 14 are available with sizes from 10nm to 5000 nm diameter, and material thicknesses of 6000 to 11000 nm.They are produced by irradiating a sheet of proprietary polycarbonatematerial. The holes 14 are fairly parallel. The material exhibits asmall degree of fluorescence, but is available dyed black. The materialstrongly absorbs ultraviolet light. The surface is specially treatedwith polyvinyl pyrrolidone to render it hydrophilic, removable bysoaking in alcohol. Depending on the density of the holes, occasionallythere is some overlap of the holes.

Gel Technology

Technically, gels are composed of an open cross-linked structure filledwith liquid. However, the term “gel” as used herein is not restricted toits strict technical definition, but rather refers to generally anymaterial that is relevant to restricting diffusion of analytes. Commongels are made from gelatin, agarose, or acrylamide. The density of theiropen cross-linked structure is easily controlled by adjusting theconcentrations of the materials used to form the gel. Some gels, such asmade from gelatin or agarose, have a melting point, below which the gelstructure forms. Other gels, such as made from acrylamide, can be formedby chemically induced polymerization or ultraviolet inducedpolymerization. These gels are heavily used in the biotechnology fieldfor separating complex mixtures of proteins, in a popular techniquecalled gel electrophoresis. In this technique, a concentrated aliquot ofprotein mixture is injected into the middle of a sheet of gel, and thenan electric field is applied to the gel at each end of the sheet. Theopen crosslinked structure of the gel is essentially a collection ofpores through which the protein molecules can pass. Since proteinmolecules typically have a characteristic charge and diameter, they willtend to migrate in the electric field at particular rates through thepores of the gel. Since each protein type has a unique charge anddiameter, the protein mixture will physically separate into itscomponents.

If the cross-linked structure of the gel is sufficiently dense, onlysmall molecules will be able to pass through the pores, and largemolecules will not be able to migrate at all from the injection point.The molecular size above which migration does not occur is commonlyreferred to as the Exclusion Limit of the gel. For polyacrylamide, theExclusion Limit can be made smaller than most typical proteins.

Chemistry

Acryamide copolymerization is a well-known reaction, commonly used togenerate gels used for gel electrophoresis. It is prepared by mixingacrylamide monomer with a cross-linking agent such asN,N′-methylenebisacrylamide (bis), and catalyzing with free radicalssuch as from persulfate anion and an initiator such as the tertiaryaliphatic airline N,N,N′,N′-tetramethylethylenediamine (TEMED). It canalso be polymerized with riboflavin and long-wavelength ultravioletlight. Additional reagents such as urea may be used to reduce theporosity of the gel. Additionally, other polymers may be included, suchas polyacrylate, to aid in lamination chemistry.

Carboxylate derivatization and amine derivatization are processes bywhich a substrate, such as a molecule or surface, is caused tochemically react with aqueous reagents, resulting in attachment ofcarboxylate or amine moieties to the substrate. For example, apolycarbonate surface may be derivatized by treatment with appropriateorganic azides to yield primary amine moieties, or its polyvinylpyrrolidone surface opened with strong base to form carboxylatemoieties. Generally, these moieties are substantially exposed to thebulk solution, where they can then be used for a variety of purposes.The chemistry used for the attachment is highly dependent on thechemistry of the substrate.

Peptide bond synthesis is a well-known reaction, commonly used forpeptide synthesis. A carboxylate moiety is treated with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to generate anunstable reactive o-acylisourea ester. This ester can react with aprimary amine moiety to form a stable amide bond. However, since thisester is so reactive, it will also react with the solvent (water) toregenerate the carboxyl moiety. This reduces the efficiency of thereaction. Improvement can be made by reacting the ester withN-hydroxysulfosuccinimide (Sulfo-NHS), which forms a semi-stableamine-reactive NHS ester. This latter ester will not react with water,but only with a primary amine moiety to form a stable amide bond.

Electrochemistry

If two electrodes are placed in an aqueous solution, and an electricpotential is applied between the electrodes, then an electric field willbe generated in the aqueous solution. Water naturally dissociatesslightly into H3O+ and OH− ions. Since these dissociation products carrya charge, they will migrate to the electrode surfaces, where the metalwill provide or extract an electron, completing a circuit of electriccurrent. Ionic salts added to the water, such as sodium chloride,introduce a much higher concentration of ions, allowing a significantlygreater flow of electric current. If a variety of ionic salts arepresent in the aqueous solution (each having chemistry that defines theease of the electron transfer process), then scanning the electrodepotential while monitoring the electric current provides acharacterization of the ionic salts in aqueous solution. Often, it isuseful in the field of electrochemistry to add an ionic salt with a verydifficult electron transfer process; such ionic salts are commonlyreferred to as “supporting electrolyte”. The structure of the electricfield at the surface of the metal electrodes is very complex, as thevarious ions form layers near the metal surface.

Chromatography

Chromatography is the physical separation of components in fluid media,followed by an appropriate detection method. Typically, a concentratedplug of as complex mixture is swept by a carrier stream (the “mobilephase”) through a narrow tube (the “column”) that has been packed withparticles (the “stationary phase”) coated with a material that weaklybinds to the components of the mixture. As the components flow past thestationary phase, some components hind more strongly than others, andwill elute out of the column slower. Hence, the eluant from the columnwill first consist of non-binding components, and then a series ofcomponents of ever-increasing binding strength. A detector that monitorssome universal characteristic of the components, such as ultravioletabsorption, indicates the presence of material as it elutes out of thecolumn. The eluant can be diverted with valves to collect eachcomponent.

In the biotechnology field, gel electrophoresis has been used widely forthe separation and detection of complex mixtures of proteins. However,the technique is somewhat unwieldy and requires fairly large volumes ofprotein. Recently, this technique is starting to he replaced by“Multidimensional Protein Identification Technology”, otherwise known asMudPIT. This technique uses a combination of columns having differentproperties, along with a set of fluidic valves, to attain proteinseparation that is superior to gel electrophoresis. The use of CapillaryZone Electrophoresis can also reduce the volume of protein used.

Matrix-Assisted Laser Desorption Ionization (MALDI)

In conventional use, MALDI involves spotting a microscopic quantity ofbiological sample onto a glass or silicon wafer, and mixing it with avaporizable compound. The wafer is dried, and then place in a massspectrometer chamber. A focused laser pulse vaporizes each sample, andthe resulting vapor ionized and accelerated through the main chamber ofa mass spectrometer. During vaporization, the biological samples areseverely fragmented, and the fragmentation pattern extracted from themass spectrometry data this procedure is widely used in industry, withequipment manufactured by Sequenom and other manufacturers.

First Embodiment Summary

A specially-constructed, layered material forms as set of reservoirsthat are loaded with a variety of spatially-separated analyte particles,and said layered material scanned with an electrochemical probe. Thisyields a map of the characteristics of the various analyte particles,which can provide useful information about biological samples.

Method in Accordance with a First Embodiment of the Invention

In a method in accordance with a first embodiment of the invention, aperforated material, such as a TEPC filter, has holes with restrictedopenings. Homogeneous or heterogeneous populations of analyte particleswithin a controlled matrix arc loaded into the TEPC filter holes, andelectrical current is used to measure the diffusion outwards, which is ameasure of presence, structural changes, and any binding interactionsinvolving the analyte particles.

The method of the invention will now be described by way of reference toFIGS. 1 to 43.

Referring to FIGS. 1A, 1B, and 2, the TEPC filter 1 has a randomdistribution of uniformly-sized holes. FIG. 1A shows the top view, andFIG. 1B is a cross sectional view. The TEPC filter 1 may have an outersurface chemically derivatized with carboxylate moieties 3. Othermaterials having similar characteristics may be substituted.

Referring to FIGS. 3, 4, and 5, a bilayer material may be formed by thefollowing process. Firstly, a dilute suspension of spheroids 4 ofuniform size is formed in a matrix of a gel precursor (such asacrylamide or melted agarose). The suspension is then applied to a thinplastic sheet 5 and wrapped around a smooth cylinder 6 that has ahydrophobic surface. The gel 7 is formed by chemical, thermal, or lightpolymerization. An example of chemical polymerization is illustrated inFIG. 4. The spacer particles 4 enforce a uniform, known thickness to thegel 7. Alter polymerization, the thin plastic sheet 5 is peeled off ofthe smooth cylinder 6, creating a bilayer material 8 consisting of a gel7 layer and as plastic 5 layer. A magnified view of this bilayermaterial is shown in FIG. 5; the spheroids 4 are not shown in thismagnified view because they are sufficiently dilute. The chemistry ofthe bilayer material 8 interface 9 is chosen such that the thin plasticsheet 5 layer may be easily removed in the future by physical orchemical means. The outer surface of the gel 7 may be chemicallyderivatized with amino moieties 10, but these are indigenous topolyacrylamide.

Alternatively to said bilayer material, a gel precursor may besandwiched between two hydrophobic smooth plates, and allowed topolymerize and dry. This forms a robust dried gel film that can beeasily handled, and then re-hydrated when needed.

Alternatively to said bilayer material, a dialysis membrane may be used,which may be purchased commercially from many vendors, such as Milliporeor Whatman.

Referring to FIGS, 6, 7, and 8, the bilayer material 8 may be chemicallybonded to the TEPC filter 1. An example of a chemical bonding mechanismis shown in FIG. 6, where carboxylate moieties and amino moietieschemically bind to form a peptide bond 11. Other chemistries may besubstituted. FIG. 7 shows the TEPC filter 1 and the bilayer material 8in close proximity, with the carboxylate derivatized surface 3 and theamino derivatized surface 10 facing each other. FIG. 8 shows the TEPCfilter 1 and the bilayer material 8 chemically bonded together with thepeptide bond 11.

Referring to FIGS. 8 and 9, the thin plastic sheet 5 may be removed byphysical or chemical means, leaving only the gel 7 layer adhered to theTEPC filter 1.

Alternatively to said chemical bonding, the gel 7 layer and the TEPCfilter I may simply be compressed together by physical force withoutchemical bonds, such as by wrapping around as cylinder and tensioningthe outer TEPC filter 1 layer.

Alternatively to said chemical bonding, the TEPC filter 1 may be placedon a surface of as conductive fluid that is immiscible with the fluidcomprising the analyte particle matrix.

There are a number of variations that are possible to the schematicshown in FIG. 9, and the examples of these are shown in FIGS. 10, 11,12, 13, 14, 15, and 16. Although the remainder of this Method will focuson the simple case of FIG. 9, it will be appreciated that thesevariations are also applicable.

Referring to FIG. 10, the outer surface of the gel 7 may be chemicallyderivatized with fluorophores 12, surfactants 13, other materials, orany combination of materials. Likewise, the inner surface of the gel 7(facing the hole 2) or the inner surface of the hole 2 may also bechemically derivatized.

Referring to FIGS. 11, 12, and 13, a spheroid 14 may be lodged in theopening of the hole 2, prior to formation of the peptide bond 11. Thisspheroid 14 would have a diameter slightly larger than the diameter ofthe hole 2. The gel 7 layer would be dimpled by the presence of thespheroid 14, creating an empty volume 15 around the spheroid 14. A force16, such as from a magnetic field gradient, electric field,gravitational field, or hydrodynamic flow, may be used to lift thespheroid 14 front its seating in the opening of hole 2, creating a smallgap 17 between the surface of the spheroid 14 and the opening of thehole 2. The empty volume 15 may be removed by brief heating of thespheroid 14 to melt the surrounding gel 7, or by saturation with gelprecursors followed by chemical polymerization. The resulting structureis shown in FIG. 13.

Referring to FIGS. 14 and 15, both ends of the hole 2 may be capped bespheroids 14. If the gel 7 is sufficiently pliable, then one of thespheroids 14 may be removed by a force, such as from a magnetic fieldgradient. This would leave a small tear 18 in the gel 7. This particularconfiguration would be especially useful for retention of materialswithin the hole 2 without an active retention mechanism.

Referring to FIG. 16, the gel may be not used, and the spheroid 14 heldin place with a force, such as from a magnetic field gradient 16.

Focusing on the simple example of FIG. 9, this structure may be used tocollect and concentrate analyte particles within the hole 2. FIG. 17 isa schematic of fluid flow 19 passing from the open end of the hole 2,through the hole 2, and then through the gel 7. Analyte particles thatare suspended within the fluid flow 19 become filtered by the gel 7, ifthe gel 7 has a sufficiently tight cross-linked structure to preventpassage. Examples of such analyte particles are proteins 20, nucleicacids, viruses, protoplasmic structures, Quantum Dots, largefluorophores 21, large electrolyte cations, large electrolyte anions,and large redox reagents. The permeability of the gel 7 may be reducedby inclusion of particles within the gel 7 during formation. Examples ofparticles that are able to pass through the gel 7 are water molecules,small electrolyte cations 22, small electrolyte anions 23, and smallredox reagents. FIG. 18 is a schematic of the net result of accumulatedanalyte particles within the hole 2.

Once there are accumulated analyte particles in the hole 2, the fluidflow 19 can be stopped. At this point, the accumulated analyte particleswill begin to diffuse outward, eventually emptying the hole 2. The fluidflow 19 may then he reinstated, and the accumulation/diffusion cyclerepeated. A gel that weakly limits diffusion may be placed at the hole 2outlet, to improve cyclability. A gel that weakly limits diffusion maybe placed within the hole, to extend the diffusion times.

During diffusion, the analyte particles in the hole 2 would havemovement pathways that can intersect with ionic particles. Since theparticles can not pass through each other, they go around each other,slowing down the movement pathways of the ionic particles. This slowingdown is dependent on the presence of analyte particles and their bindingprocesses, and thereby forms the basis of this Method.

During diffusion, the analyte particles in the hole 2 may be drivenaxially with a migration three (e.g. force resulting from an electricfield) in addition to diffusion.

This process is also applicable to the accumulation part of the cycle,but analysis is complicated by the addition of fluid flow 19 forces.

During the diffusion or accumulation parts of the cycle, the fluid flow19 may be given a high-frequency axial oscillation, fur the purpose ofmodifying particle movement. For example, the spheroid 14 of FIG. 15 mayhave a magnetic moment and be magnetically oscillated to pump analyteparticles through the tear 18. As another example, the outer (or inner)surface of the gel 7 in FIG. 9 may be subjected to pressure pulsations,causing the analyte particles within the hole 2 to likewise oscillate.

There are numerous mechanisms by which particles in the hole 2 may bydriven axially with a force in addition to diffusion. An example of oneof these mechanisms is illustrated in FIG. 22.

Referring to FIG. 22, electrolytes or redox reagents that are able totraverse the gel 7 are moved through the gel 7 by an electric fieldgenerated by Working (−W), Counter (C+), and Reference (R) electrodes.

In each of these examples for mechanisms by which particles in the hole2 may by driven axially with a force in addition to diffusion, themovement pathways themselves may be slowed down by several differentmechanisms. Examples of some of these mechanisms are illustrated inFIGS. 27 and 28.

Referring to FIG. 27, a small electrolyte cation 22 and smallelectrolyte anion 23 may be moved in as straight line by an electricfield if there are no obstacles in its path. However, if there areobstacles, such as an analyte particle 29 and an analyte particle 27,then the cations 22 and anions 23 will need to go around the analyteparticles, slowing down the ionic axial movement. If the analyteparticles have a binding interaction, they will form a large complex 28,causing the ionic axial movement to be slowed down even more. Forexample, a typical protein, such as Bovine Serum Albumin (BSA), has asize of about 4 nm×4 nm×14 nm. With a TEPC filter 1 hold 2 diameter of50 nm, a single BSA protein molecule would be blocking a significantfraction (0.8% to 2.9%) of the cross-sectional area of the hole 2, andhence significantly reduce the ionic particle flow.

Referring to FIG. 28, obstacles 29, 30, and 31 themselves may have anelectric charge. Upon application of an electric field, they mayinteract in complex ways with the movement of the electrolytes or redoxreagents.

The transmission of electrolyte molecules or redox reagents through thegel 7, dependent upon obstacle characteristics within the hole 2,provides a sensitive way to perform measurements of the obstaclecharacteristics. FIG. 31 illustrates the application of an electricfield with Working, Counter, and Reference electrodes. The ionicparticle flow will create an electric current in the electrodes that isresisted by both the obstacles and by the gel 7, but since theresistance of the gel 7 is relatively constant, this resistance can besubtracted out by cycling the measurements.

The overall apparatus in the sample loading state is shown in FIG. 32. Afluid flow of analyte particles, such as a complex sequence of proteinseluting from a chromatography column 32, is directed to flow into aparticular region of the TEPC filter 1. A lower pressure on the oppositeside causes the solvent and other small molecules to pass through theTEPC filter 1 and gel 7, accumulating analyte particles within the TEPCfilter 1 holes 2. As analyte particles are eluted from thechromatography column 32, the TEPC filter 1 surface is moved in ascanning motion. The different analyte particles that elute are trappedin spatially distinct locations within the TEPC filter 1 Following thisoperation, a second scan may be done with different analyte particleseluting from the chromatography column 32, producing a large number ofunique binary mixtures (of controllable proportions) across the area ofthe TEPC filter 1. Further scans could be used to add even morecomplexity to the populations within the holes 2. This would befunctionally equivalent to “Sandwich Array” technology, for reducing theproblem of protein cross-reactivity. It is even possible to includelipid micelles, colloids, immobilized materials, or Phage AntibodyDisplay technology within the holes 2, allowing retained analyteparticles to interact with that environment. Furthermore, it is possibleto extract analyte particles from one set of holes 2 and add it toanother set of holes 2.

The overall apparatus in the measurement state is shown in FIG. 33. Anelectrode 33, or plurality of electrodes, is scanned across the surfacesuch as the TEPC filter 1 surface, and the electric current measured.Information about the position of the electrode and the electric currentis compiled into a conductance map 34. Examples of the structure of theelectrode include an exposed metal surface surrounded by an insulator,and a tube filled with conductive fluid.

Referring to FIG. 35, the result of measurement is a set of largelyuniform conductance maps 34. Most of the area is uniformly conductivewith the exception of a few low-conductance areas having significantmigration obstacles (e.g. analyte particles). The characteristics ofthese low-conductance areas, relative to the uniformly conductive areas,is the basis of the analytical signal.

Referring to FIG. 33, restriction of the area to be measured can beachieved, by an insulating tube, filled with an electrically conductivefluid, with one end in physical contact with said sheet of material, aninsulating sheet with a hole that is applied to the surface of saidsheet of material, or an insulating water-immiscible fluid that isapplied to the surface of said sheet of material. In the latter case,upon pressurization of the gel side of the TEPC filter 1, surfacetension of water within the holes forms isolated aqueous protuberancesalong the other surface of the TEPC filter 1, that may be scanned withan insulating tube, filled with an electrically conductive fluid.

Referring to FIG. 37, the electrical conductance is graphed as afunction of time for an area that has protein present, and another areathat has no protein present. After the electric field E is actuated, thepresence of protein causes a vertical (increased) shift in the current ilevel, which can be quantified by a delta current i measurement.

Referring to FIG. 39, the theoretical results for protein present versusno protein present are compared. The electric field E is repeatedlyactuated in a cycle, yielding repeated delta current i measurements. Ona longer time scale, the hydrodynamic pressure P is also repeatedlyactuated in a cycle. When increased pressure causes protein toaccumulate, the delta current increases. When the pressure is released,the protein diffuses outward, and the delta current decreases. Cyclingthe hydrodynamic pressure thus provides a continual series of deltacurrent peaks that can be averaged for sensitive detection of thepresence of the protein.

Referring to FIG. 41, the theoretical results for protein bindingpresent versus protein non-binding are compared. Protein that binds toanother protein will have greater steric effects, becoming a largeobstacle and having a slower diffusion rate; this results in a series ofcontinual delta current i peaks that are of large amplitude. Proteinthat does not bind to another protein will have lesser steric effects,not becoming a large obstacle and not having a slower diffusion rate;this results in a series of continual delta current peaks that are ofsmall amplitude. Note that since there are multiple proteins present,this smaller amplitude may have multiple peaks.

A summary of the measurement results is shown in FIG. 43. Themeasurements provide a map of the analyte particle characteristicsacross the area of the TEPC filter 1. Certain areas 38 will havemeasurement characteristics of interest to a scientist performing themethod. The identity of the contents of these areas can then be done bya variety of techniques, such as by mass spectrometry, or by knowledgeof the chromatography system that originally delivered the contents.

Substantially the same functionality may be achieved by use of similarstructures, such as a perforated monolayer film instead of a TEPC/gelstructure, where diffusion is radial instead of axial.

Second Embodiment Summary

A specially-constructed, layered material forms a set of reservoirs thatare loaded with a variety of spatially-separated analyte particles, andsaid layered material imaged for fluorescence emission. This yields amap of the characteristics of the various analyte particles, which canprovide useful information about biological samples.

Method in Accordance with a Second Embodiment of the Invention

In a method in accordance with a second embodiment of the invention, aperforated material, such as a TEPC filter, has holes with restrictedopenings. Homogeneous or heterogeneous populations of analyte particleswithin a controlled matrix are loaded into the TEPC filter holes, andfluorescence is used to measure the diffusion outwards, which is ameasure of presence, structural changes, and any binding interactionsinvolving the analyte particles.

The method of the invention will now be described by way of reference toFIGS. 1 to 43.

Referring to FIGS. 1A, 1B, and 2, the TEPC filter 1 has a randomdistribution of uniformly-sized holes. FIG. 1A shows the top view, andFIG. 1B is a cross sectional view. The TEPC filter 1 may have an outersurface chemically derivatized with carboxylate moieties 3. Othermaterials having similar characteristics nay be substituted.

Referring to FIGS. 3, 4, and 5, a bilayer material may be formed by thefollowing process. Firstly, a dilute suspension of spheroids 4 ofuniform size is formed in a matrix of a gel precursor (such asacrylamide or melted agarose). The suspension is then applied to a thinplastic sheet 5 and wrapped around a smooth cylinder 6 that has ahydrophobic surface. The gel 7 is formed by chemical, thermal or lightpolymerization. An example of chemical polymerization is illustrated inFIG. 4. The spacer particles 4 enforce as uniform, known thickness tothe gel 7. After polymerization, the thin plastic sheet 5 is peeled offof the smooth cylinder 6, creating a bilayer material 8 consisting of agel 7 layer and a plastic 5 layer. A magnified view of this bilayermaterial is shown in FIG. 5; the spheroids 4 are not shown in thismagnified view because they are sufficiently dilute. The chemistry ofthe bilayer material 8 interface 9 is chosen such that the thin plasticsheet 5 layer may be easily removed in the future by physical orchemical means. The outer surface of the gel 7 may be chemicallyderivatized with amino moieties 10, but these are indigenous topolyacrylamide.

Alternatively to said bilayer material, a gel precursor may besandwiched between two hydrophobic smooth plates, and allowed topolymerize and dry. This forms a robust dried gel film that can beeasily handled, and then re-hydrated when needed.

Alternatively to said bilayer material, a dialysis membrane may be used,which may be purchased commercially from many vendors, such as Milliporeor Whatman.

Referring to FIGS. 6, 7, and 8, the bilayer material may be chemicallybonded to the TEPC filter 1. An example of a chemical bonding mechanismis shown in FIG. 6, where carboxylate moieties and amino moietieschemically bind to form a peptide bond 11. Other chemistries may besubstituted. FIG. 7 shows the TEPC filter 1 and the bilayer material 8in close proximity, with the carboxylate derivatized surface 3 and theamino derivatized surface 10 facing each other. FIG. 8 shows the TEPCfilter 1 and the bilayer material 8 chemically bonded together with thepeptide bond 11.

Referring to FIGS. 8 and 9 the thin plastic sheet 5 may be removed byphysical or chemical means, leaving only the gel 7 layer adhered to theTEPC filter 1.

Alternatively to said chemical bonding, the gel 7 layer and the TEPCfilter 1 may simply be compressed together by physical force withoutchemical bonds, such as by wrapping around a cylinder and tensioning theouter TEPC filter 1 layer.

Alternatively to said chemical bonding, the TEPC filter 1 may be placedon a surface of a conductive fluid that is immiscible with the fluidcomprising the analyte matrix.

There are a number of variations that are possible to the schematicshown on FIG. 9, and the examples of these are shown in FIGS. 10, 11,12, 13, 14, 15, and 16. Although the remainder of this Method will focuson the simple case of FIG. 9, it will be appreciated that thesevariations are also applicable.

Referring to FIG. 10. the outer surface of the gel 7 may be chemicallyderivatized with fluorophores 12, surfactants 13, other materials, orany combination of materials. Likewise, the inner surface of the gel 7(facing the bole 2) or the inner surface of the hole 2 may also bechemically derivatized.

Referring to FIGS. 11, 12, and 13, a spheroid 14 may be lodged in theopening of the hole 2, prior to formation of the peptide bond 11. Thisspheroid 14 would have a diameter slightly larger than the diameter ofthe hole 2. The gel 7 layer would be dimpled by the presence of thespheroid 14, creating an empty volume 15 around the spheroid 14. A force16, such as from a magnetic field gradient, electric field,gravitational field, or hydrodynamic flow, may be used to lift thespheroid 14 from its seating in the opening of hole 2, creating a smallgap 17 between the surface of the spheroid 14 and the opening of thehole 2. The empty volume 15 may be removed by brief heating of thespheroid 14 to melt the surrounding gel 7, or by saturation with gelprecursors followed by chemical polymerization. The resulting structureis shown in FIG. 13.

Referring to FIGS. 14 and 15, both ends of the hole 2 may be capped byspheroids 14. If the gel 7 is sufficiently pliable, then one of thespheroids 14 may be removed by a force, such as from a magnetic fieldgradient. This would leave a small tear 18 in the gel 7. This particularconfiguration would be especially useful for retention of materialswithin the hole 2 without an active retention mechanism.

Referring to FIG. 16, the gel may be not used, and the spheroid 14 heldin place with a force, such as from a magnetic field gradient 16.

Focusing on the simple example of FIG. 9, this structure may be used tocollect and concentrate analyte particles within the hole 2. FIG. 17 isa schematic of fluid flow 19 passing from the open end of the hole 2,through the hole 2, and then through the gel 7. Analyte particles thatare suspended within the fluid flow 19 become filtered by the gel 7, ifthe gel 7 has a sufficiently tight cross-linked structure to preventpassage. Examples of such analyte particles are proteins 20, nucleicacids, viruses, protoplasmic structures, Quantum Dots, largefluorophores 21, large electrolyte cations, large electrolyte anions,and large redox reagents. The permeability of the gel 7 may be reducedby inclusion of particles within the gel 7 during formation. Examples ofparticles that are able to pass through the gel 7 are water molecules,small electrolyte cations 22, small electrolyte anions 23, and smallredox reagents. FIG. 18 is a schematic of the net result of accumulatedanalyte particles within the hole 2.

Once there are accumulated analyte particles in the hole 2, the fluidflow 19 can be stopped. At this point, the accumulated analyte particleswill begin to diffuse outward, eventually emptying the hole 2. The fluidflow 19 may then be reinstated, and the accumulation/diffusion cyclerepeated. A gel that weakly limits diffusion may be placed at the hole 2outlet, to improve cyclability. A gel that weakly limits diffusion maybe placed within the hole, to extend the diffusion times.

During diffusion, the analyte particles in the hole 2 would havemovement pathways that can intersect with ionic or fluorescentparticles. Since the particles can not pass through each other, they goaround each other, slowing down the movement pathways of the ionic orfluorescent particles. This slowing down is dependent on the presence ofanalyte particles and their binding processes, and thereby forms thebasis of this Method.

During diffusion, the analyte particles in the hole 2 may be drivenaxially with a migration force (e.g. force resulting from an electricfield) in addition to diffusion.

This process is also applicable to the accumulation part of the cycle,but analysis is complicated by the addition of fluid flow 19 forces.

During the diffusion or accumulation parts of the cycle, the fluid flow19 may be given a high-frequency axial oscillation, for the purpose ofmodifying particle movement. For example, the spheroid 14 of FIG. 1 mayhave a magnetic moment and be magnetically oscillated to pump largeanalyte particles through the tear 18. As another example, the outer (orinner) surface of the gel 7 in FIG. 9 may be subjected to pressurepulsations, causing the analyte particles within the hole 2 to likewiseoscillate.

There are numerous mechanisms by which particles in the hole 2 may bydriven axially with a force in addition to diffusion. Examples of someof these mechanisms are illustrated in FIGS. 19, 20, 21, and 23.

Referring to FIG. 19, fluorophores that me unable to traverse the gel 7are moved towards (or away from) the inner gel 7 surface by an electricfield generated by Working (−W), Counter (C+), and Reference (R)electrodes.

Referring to FIG. 20, fluorophores that are unable to traverse the gel 7are moved towards for away from) the inner gel 7 surface by a magneticfield gradient.

Referring to FIG. 21, fluorophores that are unable to traverse the gel 7are moved towards (or away from) the inner gel 7 surface by agravitational field.

Referring to FIG. 23, large electrolyte cations 24, large electrolyteanions 25, or redox reagents that are unable to traverse the gel 7 aremoved towards (or away from) the inner gel 7 surface and the outer gel 7surface by an electric field, creating a capacitor.

The capacitor has behavior that warrants additional description in FIGS.24, 25, and 26.

Referring to FIG. 24, the electric field that is axial to the hole 2 hasa complex structure. This complex structure is analogous to the electricfield that exists near metallic electrodes in ordinary electrochemicalstudies. At a distance far away from the outer surface of the gel 7, inthe bulk solution, the electric field is small and does not changesignificantly with distance. Approaching the outer surface of the gel 7,the electrolyte exhibits an increased concentration, causing theelectric field to rise exponentially; this is commonly called theGouy-Chapman Layer. Extremely close to the outer surface of the gel 7,the electrolyte forms a double-layer of alternating charge; this iscommonly called the Helmholtz Layer. The presence of surfactantmolecules 13 may assist in the shaping of the field within the HelmholtzLayer. Continuing onward into the gel 7, the electric field subsides.Upon exiting the gel 7 on the inner surface, the electric field has astructure that mirrors the structure for the outer surface.

Referring to FIG. 25, the strong electric field in the Helmholtz layerof the outer gel 7 surface causes an electric charge, such as anelectron, to be transferred from an electrolyte (or a suitable redoxreagent) anion 25 (or cation) to a fluorophore 12 bound to the surface.After the molecule 25 transfers its charge, it becomes another molecule26. The fluorophore 12 will have its fluorescence characteristicschanged by the charge transfer. For example, fluorescein in itsuncharged state is non-fluorescent, but when negatively charged becomesintensely fluorescent.

In each of these examples for mechanisms by which particles in the hole2 may by driven axially with a force in addition to diffusion, themovement pathways themselves may be slowed down by several differentmechanisms. Examples of some of these mechanisms are illustrated inFIGS. 26, 27, and 28.

Referring to FIG. 26, a fluorophore 21 may be moved in a straight lineby a migration force (such as from an electric field, magnetic fieldgradient, or gravitational field) if there are no obstacles in its path.However, if there are obstacles, such as an analyte particle 20 and ananalyte particle 27, then the fluorophore 21 will need to go around theanalyte particles, slowing down the fluorophore axial movement. If theanalyte particles have a binding interaction, they will form a largecomplex 28, causing the fluorophore axial movement to be slowed downeven more. For example, a typical protein, such as Bovine Serum Albumin(BSA), has a size of 4 nm×4 nm×14 nm. With a TEPC filter 1 hole 2diameter of 50 nm, a single BSA protein molecule would be blocking asignificant fraction (0.8% to 2.9%) of the cross-sectional area of thehole 2, and hence significantly reduce the fluorophore flow.

Referring to FIG. 27, a large electrolyte cation 24 and largeelectrolyte anion 25 may he moved in a straight line by an electricfield if there are no obstacles in its path. However, if there areobstacles, such as an analyte particle 20 and an analyte particle 27,then the cation 24 and union 25 will, need to go around, the analyteparticles, slowing down the ionic axial movement. If the analyteparticles have a binding interaction, they will form a large complex 28,causing the ionic axial movement to be slowed down even more.

Referring to FIG. 28, obstacles 29, 30, and 31 themselves may have anelectric charge. Upon application of an electric field, they mayinteract in complex ways with the movement of the electrolytes or redoxreagents.

The accumulation or activation of fluorophores in the vicinity of thegel 7, depending upon obstacle characteristics within the hole 2,provides a sensitive way to perform measurements of the obstaclecharacteristics. FIGS. 29 and 30 illustrate the application ofultraviolet light to the fluorophores, and the resulting emitted visiblelight. The intensity of the emitted visible light is dependent upon theconcentration of active fluorophores in the vicinity of the gel 7.Fluorophores that are located at a distance down the hole 2 will notfluoresce, because the polycarbonate material of the TEPC filter 1 isstrongly absorbing for ultraviolet light. Note that although a primaryadvantage of this method is to avoid the need to label an analyteparticle with a fluorophore, it is within the scope of this method toinclude analyte particles labeled with fluorophores.

The overall apparatus in the sample loading state is shown in FIG. 32. Afluid flow of analyte, such as a complex sequence of proteins elutingfrom a chromatography column 32, is directed to flow into a particularregion of the TEPC filter 1. A low pressure on the opposite side causesthe solvent and other small molecules to pass through the TEPC filter 1and gel 7, accumulating analyte particles within the TEPC filter 1 holes2. As analyte particles are eluted from the chromatography column 32,the TEPC filter 1 surface is moved in a scanning motion. The differentanalyte particles that elute are trapped in spatially distinct locationswithin the TEPC filter 1. Following this operation, a second scan may bedone with different analyte particles cluing from the chromatographycolumn 32, producing a large number of unique binary mixtures (ofcontrollable proportions) across the area of the TEPC filter 1. Furtherscans could be used to add even more complexity to the populationswithin the holes 2. This would be functionally equivalent to “SandwichArray” technology, for reducing the problem of protein cross-reactivity.It is even possible to include lipid micelles, colloids, immobilizedmaterials, or Phage Antibody Display technology within the holes 2,allowing retained analyte particles to interact with that environment.Furthermore, it is possible to extract analyte particles from one set ofholes 2 and add it to another set of holes 2.

The overall apparatus in the measurement state is shown in FIG. 34. Abeam of fluorescence excitation light, such as ultraviolet light, isdirected towards a surface such as the gel 7 surface. The fluorescenceemission light, such as visible light, is collected by a lens 35 orlight pipe, and a photosensor 36, such as a digital camera. Informationfrom the camera is compiled into a photographic image 37.

Referring to FIG. 36, the result of measurement is a series of changingphotographic images 37 when the migration force (such as from anelectric field, magnetic field gradient, or gravitational field) isapplied. Initially, the photographic image is relatively dark. After abrief amount of time, most of the area is fluorescent with the exceptionof a few delayed areas having significant migration obstacles (e.g.analyte particles). Soon, however, as migration completes in thesedelayed areas, the whole area of the image becomes uniformlyfluorescent. The temporal characteristics of these delayed areas,relative to the un-delayed areas, is the basis of the analytical signal.

Referring to FIG. 38, the fluorescence is graphed as a function of timefor an area that has protein present, and another area that has noprotein present. After the electric field E is actuated, the presence ofprotein causes a horizontal (temporal) shift in the sigmoidal curve,which can be quantified by a delta time t measurement.

Referring to FIG. 40, the theoretical results for protein present versusno protein present are compared. The migration force is repeatedlyactuated in a cycle, yielding repeated delta time t measurements. On alonger time scale, the hydrodynamic pressure P is also repeatedlyactuated in a cycle. When increased pressure causes protein toaccumulate, the delta time increases. When the pressure is released, theprotein diffuses outward, and the delta time decreases. Cycling thehydrodynamic pressure thus provides a continual series of delta timepeaks that can be averaged for sensitive detection of the presence ofthe protein.

Referring to FIG. 42, the theoretical results for protein bindingpresent versus protein non-binding are compared. Protein that binds toanother protein will have greater steric effects, becoming a largeobstacle and having a slower diffusion rate; this results in a series ofcontinual delta time t peaks that are of large amplitude. Protein thatdoes not bind to another protein will have lesser steric effects, notbecoming a large obstacle and not having as slower diffusion rate; thisresults in a series of continual delta time peaks that are of smallamplitude. Note that since there are multiple proteins present, thissmaller amplitude may have multiple peaks.

A summary of the measurement results is shown in FIG. 43. Themeasurements provide a map of the analyte particle characteristicsacross the area of the TEPC filter 1. Certain areas 38 will havemeasurement characteristics of interest to a scientist performing themethod. The identity of the contents of these areas can then be done bya variety of techniques, such as by mass spectrometry, or by knowledgeof the chromatography system that originally delivered the contents.

Substantially the same functionality may be achieved by use of similarstructures, such as a perforated monolayer film instead of a TEPC/gelstructure, where diffusion is radial instead of axial.

Third Embodiment Summary

A specially-constructed, layered material forms a set of reservoirs thatare loaded with a variety of spatially-separated analyte particles, andsaid layered material sampled by mass spectrometry. This yields a map ofthe compositions of the various analyte particles, which can provideuseful information about biological samples.

Method in Accordance with a Third Embodiment of the Invention

In a method in accordance with a third embodiment of the invention, theholes of a TEPC filter (or functionally equivalent structure) arerestricted at the openings. Homogeneous or heterogeneous populations ofanalyte particles within a controlled matrix are loaded into the TEPCfilter holes, and the hole ends are tightly capped by spheroidsforcefully corked into the hole ends. The assembly is loaded into avacuum chamber, targeted by a laser beam, and the resulting vapordirected into a mass spectrometry chamber. Ionization and mass/chargedetection provides a means of identification of the analyte particles.

The method of the invention will now be described by way of reference toFIGS. 44 to 48.

Referring to FIG. 44, a composition of analyte particles 20, 22, and 23is enclosed within the hole 2 of TEPC filter 1, by having a spheroid 14at each end of the hole 2.

Referring to FIGS. 45 and 46, tightly squeezing the TEPC filter 1between two smooth rollers to exert as compressive force 39 would causeeach spheroid 14 to stopper both ends of the hole 2. The TEPC filter 1could then be removed from the preparatory apparatus, yielding a stablematerial shown in FIG. 46 that contains the analyte particles.

Referring to FIG. 47, the stable material is put into a vacuum chamber.The aqueous matrix containing the analyte particles within the hole 2 isprotected from the vacuum by the spheroids 14 stoppering the hole 2 andsealing in the aqueous matrix. Additional sealing may be provided with athin polymer film overcoat. An intense, focused laser beam 40 is thendirected at the hole 2.

Referring to FIG. 48, the laser beam would vaporize the upper stopper ofthe TEPC filter 2 hole 2, causing the aqueous matrix to explode outwardsinto the vacuum, dispersing it as a gas. This is analogous tomatrix-assisted laser desorption ionization (MALDI) analysis. However,the biological sample receives much less thermal stress, leading to aless complex fragmentation pattern. This reduced complexity may beanalytically useful.

Summary

The scope of these Methods covers the tasks of detection,characterization, and identifying of analyte panicles, and thecharacterization of any interactions involving them, using theproperties of nanoscale reservoirs.

Current methods for performing these tasks suffer from a variety ofdeficiencies, such as the interference from labeling techniques andcross-reactivity. These deficiencies are eliminated. Furthermore, themethods are useful for analysis of extremely low concentrations ofanalyte particles.

A specific field where these Methods would be useful is inbiotechnology, for the measurement of protein interactions. There are anextremely large number of proteins used in every biological system,which interact in a complex network that is dependent on many factors.Diseases distort this network, adding or removing components andinteraction pathways. An understanding of these systems allows earlydiagnosis of disease, and a way to chemically repair the system throughdrug therapy. The most populous and stable proteins within these systemshave been partially studied, but much further study is warranted. Theaddition of new and more powerful tools, such as the Methods describedherein, to the repertoire of medical researchers would deepen theunderstanding of the protein networks and allow the development of newdrug therapies.

The term “particles” as used herein includes molecules, cells,multicellular structures, subcellular components, viruses, prions,proteins, polymers, ions, colloids, and fluorophores. The particles maybe suspended or dissolved. The particles do not necessarily need to bebiologically relevant.

The term “analyte” as used herein describes particles that are to bemeasured.

The term “gel” as used herein is not restricted to its strict technicaldefinition, but rather includes generally any material that is relevantto restricting diffusion of analyte particles. The term “gel” isintended to include, but is not limited to, gelatin, agarose,polyacrylamide, polyacrylate, permeable polymers, permeable copolymers,starch, aerogel, collodion, dialysis membrane, immiscible fluid, any ofthe above-listed materials in a chemically modified form, any of theabove-listed materials embedded with particles, and any combination ofthe above listed materials.

The term “immiscible fluid” as used herein includes fluids that areimmiscible with the analyte particle matrix.

The term “large” as used herein describes those particles that can notpass through the restriction at the end of the TEPC filter hole, such asby the gel. The term “small” as used herein describes those particlesthat can pass through the restriction at the end of the TEPC filterhole, such as by the gel.

The term “force” as used herein includes a force resulting from anelectric, field, a force resulting from a magnetic field gradient, aforce resulting from a gravitational field, centripetal force,centrifugal force, force resulting from hydrodynamic pressure, a forceresulting from hydrostatic pressure, or a combination of such forces.

Electric fields may be generated capacitively, without direct electrodecontact with ionic or redox species. Multiple sets of electrodes may beused as needed to achieve the necessary electric fields. For example,one set of electrodes may be used to exert strong migration forces,while another set of electrodes is used for analyte particlemeasurement.

Measurement of electric current may be structured to constituteresistance, conductance, impedance, capacitance, and inductancemeasurements, and combinations thereof. For example, characterization oflipid micelles, whole cells, or other materials with impedanceboundaries may be assisted by capacitance measurements, andcharacterization of chiral analytes may be assisted by inductancemeasurements.

Analyte particles may be delivered to the apparatus by local rupture ofintact cells, or by other techniques, in addition to standardchromatographic techniques.

The holes or perforations in said TEPC material, or its functionalequivalent, may be sized for close fitting of individual cells, so thatelectrical impedance and capacitance are determined largely through thebulk of the cell.

The methods described herein may be combined with conventionalmicrochannel array technology, commonly referred to as “lab-on-a-chip”technology.

While the present invention has been described with reference to certainpreferred embodiments, one of ordinary skill in the art will recognizethat other additions, deletions, substitutions, modifications, andimprovements can be made while remaining within the spirit and scope ofthe present invention as defined by the claims.

1-20. (canceled)
 21. A method for analyzing an analyte, comprising: (a).providing a sheet of material having a plurality of through holes, thethrough holes having openings on both faces of the sheet of material;(b). restricting openings of the through holes on at least one face ofthe sheet of material; (c). inserting an analyte into the through holes,wherein passage of the analyte through the through holes is restrictedby the restricting in step (b); (d). passing a migration force axiallythrough the through holes containing the analyte; and (e). measuring achange in fluorescence with time, wherein the change is indicative ofthe diffusion rates of the analyte diffusing out of the through holes.22. The method of claim 21, wherein the analyte is a fluorescentanalyte.
 23. The method of claim 21, wherein the analyte is labeled witha fluorescent molecule or material.
 24. The method of claim 23, whereinthe fluorescent material comprises quantum dots.
 25. The method of claim21, wherein a fluorescent molecule or material is provided in thethrough holes, and the analyte is not labeled with the fluorescentmolecule or material.
 26. The method of claim 21, wherein the sheet ofmaterial comprises a material having a property selected from the groupconsisting of: being electrically insulating; being chemically inert;having a thickness in the range of 500 nm to 1,000,000 nm; having athickness in the range of 1 nm to 10 cm; having through holes ofdiameters in the range of 10 nm to 5,000 nm; having through holes ofdiameters in the range of 1 nm to 1 cm; having through holes with aninner surface that is chemically derivatized; having an outer surfacethat is chemically derivatized; and having through holes filled with agel.
 27. The method of claim 21, wherein the sheet of material isselected from the group consisting of: a polycarbonate; a track-etchedpolycarbonate; a polymer drilled with a plurality of holes; a polymerchemically etched with a plurality of holes; a glass drilled with aplurality of holes; a glass chemically etched with a plurality of holes;a perforated polymer film; a perforated monolayer film; and a perforatedmultilayer film.
 28. The method of claim 21, wherein the restricting instep (b) comprises restricting the through hole openings by a layer of agel in contact with a surface of the sheet of material.
 29. The methodof claim 28, wherein the gel comprises a material selected from thegroup consisting of: a gelatin; an agarose; a polyacrylamide; apolyacrylate; a permeable polymer; a permeable copolymer; a starch; anaerogel; a collodion; and a dialysis membrane.
 30. The method of claim28, wherein the gel is immiscible with the analyte.
 31. The method ofclaim 21, wherein the restricting in step (b) comprises restricting thethrough hole openings using spheroids, and wherein diameters of thespheroids are sufficient to cause restriction of fluid flow through thethrough holes.
 32. The method of claim 31, wherein the spheroids arecompressed into the through hole openings.
 33. The method of claim 31,further comprising holding the spheroids in position relative to thethrough hole openings using a gel matrix.
 34. The method of claim 33,further comprising removing the spheroids from the gel matrix.
 35. Themethod of claim 31, further comprising holding the spheroids in positionrelative to the through hole openings by an electric field, a magneticfield gradient, a gravitational field, a centripetal force, acentrifugal force, hydrodynamic pressure, hydrostatic pressure, chemicalbonding, or a combination thereof.
 36. The method of claim 21, whereinthe migration force is selected from the group consisting of: a forceresulting from an electric field; a force resulting from a magneticfield gradient; a force resulting from a gravitational field; acentripetal force; a centrifugal force; a force resulting fromhydrodynamic pressure; a force resulting from hydrostatic pressure; andcombinations thereof.
 37. The method of claim 21, wherein the analyte isconcentrated in the through holes.
 38. The method of claim 21, furthercomprising reinserting the analyte into the through holes after theanalyte diffuses out of the through holes, and repeating steps (c)-(d).39. The method of claim 21, wherein the through holes are a first set ofthrough holes, and wherein the analyte is extracted from the first setof through holes and added to a second set of through holes.
 40. Themethod of claim 21, wherein the change in fluorescence with time ismeasured by a photometric system capable of measuring the fluorescenceof a selected area of the sheet of material.